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•Thanks to
• Sleeman Brewery, Guelph Ontario for the use of their
laboratories to carry out this work.
• Jonathan Crawshaw, Sleeman Brewery, Guelph, Canada, for
facilitating this work.
• Bart Schuurman Hess, Sealedair, Food Care, Oakville, Ontario,
Canada for his enthusiasm for the work, and bringing us all
together.
1
Acknowledgements
Pitching yeast is reported to act as a reservoir for low levels of bacterial contamination
Briggs 2004
Yeast Washing
Need to determine if the
brewery is going to adopt yeast
washing as a part of its brewing
strategy
3
Yeast Washing
•The strategies:
a. Wash all yeast
b. Wash only when problems are evident
c. Not wash at all
• We will be addressing the first two strategies
4
Yeast Washing
•Yeast “purification” with Louis Pasteur
Around 1876
Found by lowering pH of yeast, the
accompanying bacteria declined in number
It is reported - He grew the yeast in acidified
cane sugar for two generations
Hind 1937
5
Yeast Washing Background
•Following Pasteur, others used an
acidified wort process – serial
tanks involved
•Using 0.1% Tartaric acid
•pH reduced to approximately 3.9
•Reduction in yeast infection
reported
6
Yeast Washing Background
•Yeast washing has evolved into the
following methods:
1. Distilled or sterile water wash
2. Acid wash
3. Acid wash with ammonium persulfate
4. (Antibiotics?) 7
Yeast Washing Background
•Antibiotics?
There use was proposed in the late 1940s and early 1950s
o Tyrothricin and Polymyxin B
It was quick realized that to use antibiotics was irresponsible and
the use of antibiotics was never done.
•Nisin?
A small polypeptide was used and accepted by the dairy industry
o Research showed it as having limited use in the brewing industry
and it has never found favour
Briggs 2004
8
Yeast Washing Background
•Yeast washing has evolved into the
following methods:
1. Distilled or sterile water wash
2. Acid wash
3. Acid wash with ammonium persulfate
9
Yeast Washing Background
• Large volumes of sterile cold hard water
•Mixed with yeast slurry
•Yeast is allowed to settle out
•Water is decanted off the yeast
The theory is the bacteria and dead cells will be
removed with the water – dilute out the bacteria
and dead cells
•This is repeated 2 or 3 times in the process
Hardwick 1995
10
Yeast Washing Using Sterile Water
• The most common method
• Many acids are reported to be used: phosphoric, citric, tartaric, sulphuric
Most common acid is food grade phosphoric acid
• Yeast slurry is acidified with dilute acid (10%) to a pH of 2.3 ± 0.1
• The acidified slurry is slowly mixed for approximately 2 hours and kept at refrigeration temperatures of 4oC
McCabe 1999
11
Acid Washing Yeast
•Criteria for acid washing yeast:
• Use food grade acid
• Wash yeast as a beer or water slurry
• Chill yeast and acid to less than 5oC
• Stir slowly and constantly
• Stir throughout the washing process
• Maintain temperature at less than 5oC
• Monitor the pH
• Do not wash more than two hours
• Pitch yeast immediately after washing McCabe 1999
12
Acid Washing Yeast
•Effectiveness of Acid washing:
• Reported that aerobic contaminants are removed
• Reported that the anaerobic beer spoilage organisms
are more resistant
• Reported that wild yeast are unaffected
Goldammer, 2008
13
Acid Washing Yeast
•More effective than acid washing
• 0.75% Ammonium Persulphate added to the acid
washing process
•Contact time is a maximum of one hour prior to
pitching
•Reported to be harder on the pitching yeast
14
Acid Washing Yeast with Ammonium
Persulphate
•The three strategies are used but have been
found to not be totally effective against the
anaerobic beer spoilage bacteria
•The work was to investigate a new yeast
washing protocol:
Chlorine Dioxide
15
Next steps
• Chlorine Dioxide is a strong but selective oxidizing agent
• Chlorine dioxide is effective over a wide pH range (2-10)
• Does not react with poly phenols (tannins), that can leave a taste in the parts per trillion
• Does not produce halogenated methane (CHX3)
Masschelein J.W.
16
Why Chlorine dioxide?
• Cannot be shipped or stored for long periods and has to be generated on site
• Generation requires mixing a strong acid with a solution of sodium chlorite in the right proportion and for the requisite time or some well known variation
• Requires specialized equipment for safe generation
• Requires careful handling as chlorine dioxide gas can escape form solution and easily exceed safety limits in the air
17
Disadvantages of chlorine dioxide
ACID CHLORITE REACTION
Two pumps mix solutions of
sodium chlorite and a strong
acid -- hydrochloric acid.
Reaction occurs in a
chamber typically 10
minutes residence time
Reaction is stoichiometric
and 99% complete if the
chemicals are in right
concentration.
To Storage Tank
or Process
5 NaCl02 + 4 HCl 4 Cl02 + 5 NaCl + 2H20
Generator Cl02 Solution
7.5%
NaCl02
9%
HCl
Schematic Acid/Chlorite Generation of
Cl02
• Not much reported in the literature
• Paper tried using activated chlorine dioxide solution mixture at pH 3
using a nominal 50ppm chlorine dioxide Johnson D., (1998)
• Mode of generation used did not specify time and used a weak acid
with chlorite
• Produces indeterminate solution mixture where precise
concentration of chlorine dioxide is not known
• Need to start with known concentration of chlorine dioxide for control
19
Will chlorine dioxide eliminate
bacteria without damaging yeast?
20
The test procedure used to challenge the bacteria infecting the yeast is
outlined in Figure1:
1. Lager yeast (Saccharomyces carlsbergensis) slurry sample was
obtained from an actual pitching tanks in a brewery.
2. 5.0ml of lactobacillus sp inoculum added to the yeast.
3. A sample of the 2000ppm solution of chlorine dioxide from a Prominent
Generator was measured for its chlorine dioxide content.
4. A calculated volume of the nominal 2000ppm chlorine dioxide solution
is added to 100.0 ml of the inoculated yeast to obtain a desired initial
concentration e.g. 100 ppm. The concentration of the 2000ppm +/-
100ppm chlorine dioxide solution was checked using the Hach DPD
method kit for chlorine dioxide #58700-51.
5. Time is started
6. Samples of the yeast are plated using a Mann-Rogosa-Sharpe (MRS)
medium for anaerobes and incubated at 28oC. Samples of the yeast
were also plated on Universal Beer Agar (UBA), and incubated at 28oC .
The aerobic bacteria were counted after 3days and the anaerobic after
5 days.
7. The viability of the yeast was determined using 0.2% Eosin Y stain
[McCaig,R].
21
H Yeast 500 mL Lactobacillus sp 5 mL
100 ppm (5.3 mL)
175 ppm (11.1 mL)
250 ppm (25 mL)
0.5 ml
Plated on MRS and UBA using S/T technique using 1/10 dilution 15 - 20 9mL dilution blanks 15 - 20 MRS/UBA plates
1
2
5
4 Chlorine Dioxide 2000 ppm
1. Lager yeast (Saccharomyces
carlsbergensis) slurry sample
was obtained from an actual
pitching tanks in a brewery.
2. 5.0ml of lactobacillus spa
inoculum added to the yeast.
4. A calculated volume of 2000ppm ClO2
added to each yeast vial to achieve
desired concentration. (5 mins apart)
Mixed gently. Time started
3 Divided into 3 X100ml samples.
5. Samples from
the different
treatments
were plated at
15 minutes
intervals.
3 100 ml in each
TABLE 1 – RESULTS FROM EXPERIMENT 1
22
• Table 1 shows the results for the first experimental attempt
• In this experiment the chlorine dioxide starting
concentrations tested were 25ppm and 100ppm
• Table 1 top window shows the initial anaerobe and aerobe
bacterial concentrations and the initial yeast viability
• The lower window shows the variation of the bacterial
concentration with time after exposure to the chlorine
dioxide
• The subsequent slides provide comments on the results in
Table 1
Starting Conditions Aerobic
[UBA(C)] Anaerobic [MRS(C)] Viability (%)
Yeast as collected (Lager CY-3) Tank
204 0 0 85
Bacteria mixture TNTC TNTC
Yeast plus bacteria TNTC TNTC
Contact
time (min)
Concentration of chlorine dioxide (ppm) Viability (%)
25 100 25ppm 100ppm
Aer Anaer Aer Anaer
0
TNTC
TNTC
TNTC
TNTC
82
82
15
TNTC
TNTC
0
500
74
74
30
TNTC
TNTC
0
250
74
72
45
TNTC
TNTC
0
75
75
77
60
TNTC
TNTC
0
45
79
71
90
TNTC
TNTC
0
0
68
74
TABLE 1
Starting Conditions Aerobic
[UBA(C)] Anaerobic [MRS(C)] Viability (%)
Yeast as collected (Lager CY-3) Tank
204 0 0 85
Bacteria mixture TNTC TNTC
Yeast plus bacteria TNTC TNTC
Contact
time (min)
Concentration of chlorine dioxide (ppm) Viability (%)
25 100 25ppm 100ppm
Aer Anaer Aer Anaer
0
TNTC
TNTC
TNTC
TNTC
82
82
15
TNTC
TNTC
0
500
74
74
30
TNTC
TNTC
0
250
74
72
45
TNTC
TNTC
0
75
75
77
60
TNTC
TNTC
0
45
79
71
90
TNTC
TNTC
0
0
68
74
TABLE 1 Chlorine dioxide at 100ppm can eliminate both the anaerobic and aerobic bacteria. The anaerobic bacterial load was TNTC, which is not a typical situation, and it took around 90mins to reduce the anaerobic bacteria to zero, and 15mins to eliminate the aerobic bacteria.
Starting Conditions Aerobic
[UBA(C)] Anaerobic [MRS(C)] Viability (%)
Yeast as collected (Lager CY-3) Tank
204 0 0 85
Bacteria mixture TNTC TNTC
Yeast plus bacteria TNTC TNTC
Contact
time (min)
Concentration of chlorine dioxide (ppm) Viability (%)
25 100 25ppm 100ppm
Aer Anaer Aer Anaer
0
TNTC
TNTC
TNTC
TNTC
82
82
15
TNTC
TNTC
0
500
74
74
30
TNTC
TNTC
0
250
74
72
45
TNTC
TNTC
0
75
75
77
60
TNTC
TNTC
0
45
79
71
90
TNTC
TNTC
0
0
68
74
TABLE 1 Yeast viability was reduced from 80% to 74% in the first 15 minutes. Yeast viability did not decrease any further after the first 15minutes
TABLE 2 – RESULTS FROM EXPERIMENT 2
26
• Table 2 shows the results for the second experimental
attempt
• In this experiment the chlorine dioxide starting
concentrations tested were 100ppm and 250ppm
• Table 2 top window shows the initial anaerobe and aerobe
bacterial concentrations and the initial yeast viability
• The lower window shows the variation of the bacterial
concentration with time after exposure to the chlorine
dioxide
• The subsequent slides provide comments on the results in
Table 2
Contact time
(min)
Concentration of chlorine dioxide
(ppm)
Viability (%) pH
100 250 100 250 100 250
Aer Anaer Aer Anaer
0
TNTC
115
TNTC
115
93
93
4.47
4.47
15
0
0
0
0
80
80
30
0
0
0
0
75
78
45
0
0
0
0
83
80
60
0
0
0
0
80
78
90
0
0
0
0
80
78
120
0
0
0
0
84
75
3.31
2.37
Starting Conditions Aerobic [UBA(C)] Anaerobic [MRS(C)] Viability (%)
Yeast as collected (Lager H) Tank
202 0 0 93
Bacteria mixture TNTC TNTC
Yeast plus bacteria TNTC 115
TABLE 2
Contact time
(min)
Concentration of chlorine dioxide
(ppm)
Viability (%) pH
100 250 100 250 100 250
Aer Anaer Aer Anaer
0
TNTC
115
TNTC
115
93
93
4.47
4.47
15
0
0
0
0
80
80
30
0
0
0
0
75
78
45
0
0
0
0
83
80
60
0
0
0
0
80
78
90
0
0
0
0
80
78
120
0
0
0
0
84
75
3.31
2.37
Starting Conditions Aerobic [UBA(C)] Anaerobic [MRS(C)] Viability (%)
Yeast as collected (Lager H) Tank
202 0 0 93
Bacteria mixture TNTC TNTC
Yeast plus bacteria TNTC 115
TABLE 2 The experiment was repeated under a more realistic initial bacterial load in experiment 2. Both aerobic and anaerobic bacteria were eliminated after the first 15mins at 100ppm
Contact time
(min)
Concentration of chlorine dioxide
(ppm)
Viability (%) pH
100 250 100 250 100 250
Aer Anaer Aer Anaer
0
TNTC
115
TNTC
115
93
93
4.47
4.47
15
0
0
0
0
80
80
30
0
0
0
0
75
78
45
0
0
0
0
83
80
60
0
0
0
0
80
78
90
0
0
0
0
80
78
120
0
0
0
0
84
75
3.31
2.37
Starting Conditions Aerobic [UBA(C)] Anaerobic [MRS(C)] Viability (%)
Yeast as collected (Lager H) Tank
202 0 0 93
Bacteria mixture TNTC TNTC
Yeast plus bacteria TNTC 115
TABLE 2 Yeast viability was reduced from 93% to 80% in the first 15 minutes. Yeast viability did not decrease any further after the first 15minutes.
Chlorine dioxide presumably eliminates any weak yeast cells. Any remaining yeast cells resist being killed by further exposure to chlorine dioxide.
30
Conclusions
• Chlorine dioxide at 100ppm appears to be specific in eliminating anaerobic
and aerobic bacteria from yeast
• The viability of the yeast does not continue to decrease with time after a
small drop in the first 15 minutes. This indicates that the yeast can survive
the action of chlorine dioxide
• The process of washing recovered yeast with chlorine dioxide takes
considerable less time, 15 - 30 minutes, than the classical acid wash
or acid-persulfate wash at 2 or more hours
• More work is required to determine the minimum effective concentration of
chlorine dioxide between 25ppm and 100ppm
• Based on these observations the washing of yeast with chlorine dioxide
merits further investigation to work out the minimum concentration of chlorine
dioxide and the details of carrying it out in a practical and safe manner
• [1] Priest F. G. and Stewart G., Handbook of Brewing (2006) 2nd ed., pp. 318-320. CRC Press Taylor and Francis Group
• [2] Bah, S. and Mckeen W. E., (1965) Beer spoilage bacteria and their control with phosphoric acid – ammonium persulphate wash. Canadian Journal of Microbiology, 11(2):309-318
• [3] Masschelein, W. J. (1979) In: Chlorine Dioxide: Chemistry and Environmental Impact of Oxychlorine Compounds pp.153-163. Ann Arbor science Publishers, Ann Arbor, MI.
• [4] Johnson D., (1998). Coming Clean: A new method of washing yeast with chlorine dioxide, The New Brewer, Sept/Oct.
• [5] McCaig, R. (1990), Evaluation of the fluorescent dye 1-anilino-8-naphthalene sulfonic acid for yeast viability determination. J. Am. Soc. Brew. Chem. 48 pp.22-25
31
References
• [6] Hind, H. L., (1937) Brewing Science and Practice,
Volume 2, Brewing Process. 4th Ed. Chapman & Hall.
London. pp. 803-4.
• [7] Goldammer, T., (2008) The Brewer’s Handbook.
Apex Publishers. pp. 82-4
• [8] Briggs, D. E., Boulton, C. A, et al, (2004) Brewing
Science and Practice, Woodhead Publishing Limited.
pp. 36-7.
• [9] Hardwick, W. A., (1995) Handbook of Brewing.
Marcel Dekker Inc. pp. 193-4
• [10] McCabe, J. T., et al. (1999) The Practical Brewer
3rd Ed., Master Brewers of the Americas. pp. 292-3.
32
References continued